In vitro Random Mutagenesis

Alanine scanning, a method to replace systematically each residue in turn by alanine, has been successful in identifying amino acid residues involved in receptor-ligand binding. However, predictions of the roles of individual amino acid residues in antigen binding can often be fraught with difficulty. Mutation of the heavy chain aspartate 101 of the monoclonal antibody to hen egg lysozyme, HyHel to alanine resulted in a 9000-fold drop in affinity, despite it not being involved in H-bonding or salt bridges (Lavoie et al., 1992). It was suggested that this residue was important for the conformation of CDR3.

Random mutagenesis of contact residues should be performed simultaneously so that they may co-vary. Additivity principles suggest that the sum of the free-energy effects for residues in contact may not equal that of the combined mutant because these side-chains can interact.

It would be expected that residues affecting the tightness of binding would be found predominantly at the interface surface of the antibody which contacts antigen. However, in growth hormone a few of the buried side chains were found, by alanine scanning, to enhance binding (Cunningham and Wells, 1989; Fuh et al., 1992). The change in affinity was due to a slowing of the offrate of the hormone (60-fold) and an increase in the on-rate (fourfold).

Gram et al. (1992) used error-prone PCR (Leung et al., 1989) to introduce random mutations into naive Fab V-region genes to achieve up to a 30-fold increase in affinity of binding to antigen. This high level of error in PCR amplification can be brought about by high cycle numbers, by unusually high or low levels of one nucleotide, by adding manganese, or by changing the magnesium concentrations. PCR mutagenesis can produce about two mutations per V gene, but it is not truly random. It has equal numbers of transitions and transversions, but G/C or C/G changes are rare.

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